Gluten is a set of storage proteins of cereals. Gluten proteins from wheat, barley, rye and probably oats, are not tolerated by genetically predisposed individuals with celiac disease (CD). In wheat, gluten is composed of an ethanol-soluble fraction (prolamins: α, β, γ and ω-gliadins) and other insoluble, glutenins (high and low molecular weight subunits) (Wieser, 2007 Food Microbiol., 24:115-119; Fasano, 2009, Sci Am, 301:54-61). Gliadins and glutenins are also unusually rich in proline (˜15%) and glutamine (˜35%) residues. As a result, while most dietary proteins are digested by gastrointestinal proteases to single amino acids, dipeptides or tripeptides, gluten proteins are not completely digested (Erickson and Kim, 1990, Annu Rev Med 41:133-139, Gray, 1991, New York: Oxford University, pp. 411-420; Ganapathy et al., 2006, Academic Press, pp. 1667-1692). Therefore, some of the gluten peptides generated during gastrointestinal digestion are highly resistant to digestion by gastric and pancreatic enzymes, so that they persist in the gut. These peptides are capable of being internalized into the intestinal cells and, therefore, glutamine residues can be deaminated by tissue transglutaminase (tTG). Genetic predisposition of individuals with CD makes them intolerant to these peptides because their immune system reacts pathologically against autoantigens generated by gluten peptides/tTG-interaction (Korponay-Szabó et al., 2007, BMJ, 335:1244-1247; Bethune and Khosla, 2008, PLoS Pathogens, 4: e34; Jabri and Sollid, 2009, Nat Rev Immunol., 9:858-870). Deamidated peptides induce an immune response mediated by T cells that causes chronic inflammation of the small intestine. Intestinal villi are destroyed due to the immunological reaction, resulting in a reduction of the intestinal absorption which can lead to symptoms such as diarrhea, anemia, stunting, weight loss, bone disorders, neurological disorders, cancer, etc. (Alaedini and Green, 2005, Ann Int Med, 142:289-299; Catassi and Fasano, 2008, Curr Opin Gastroenterol., 24:687-691; Tack et al., 2010, Gastroenterol Hepatol., 7:204-213).
One of the main gluten peptides described to date is the 33-mer peptide from α2-gliadin (Shan et al., 2002, Science, 297:2275-2279; Bethune et al., 2009, Chem Biol, 16:868-881) that has been shown to be resistant to gastrointestinal digestion, substrate of the tTG mediated deamination and highly reactive with T cells isolated from celiac patients. The identification of the 33-mer peptide and other peptides, helps to demonstrate that gluten epitopes with high antigenicity are located in gliadin regions rich in proline and glutamine residues (Shan et al., 2002, Science, 297: 2275-2279; Tye-Din et al., 2010, Sci Transl Med 2:41 RA51).
Nowadays, the only existing therapy for patients with celiac disease is a strict gluten-free diet (GFD). Non-compliance with the GFD has been associated with osteoporosis, iron deficiency anemia, depression and infertility, all of which is improved, to some extent, by adhering to the gluten-free diet. These observations give us an idea of the importance of adherence to a GFD to reduce symptoms, prevent nutritional deficiencies and improve the quality of life of these patients. However, several studies based on intestinal biopsies have suggested that dietary transgressions are relatively frequent, being between 32.6% and 55.4% in the populations studied (Ciacci et al., 2002, Digestion, 66: 178-185; Sylvester and Rashid, 2007, Can J Gastroenterol., 21:557-564). The lack of adherence to a strict gluten-free diet is the main reason for poorly controlled celiac disease in adults.
In addition, there is a part of the celiac population that does not seem to respond positively to the GFD and suffer symptoms of persistent or recurrent malabsorption and intestinal villous atrophy. This population could be suspected of having refractory CD, a rare disease (approximately 5%-10% of patients with CD) that appears in patients without apparent positive response to the gluten-free diet (Al-Shot et al., 2007, Dig Dis 25:230-236, Freeman, 2009, Gut Liver, 3:237-246; Rubio-Tapia and Murray, 2010, Gut, 59:547-557). Although this refractory disease was described in patients with assumed total absence of gluten intake, involuntary ingestion and hypersensitivity to a small amount of gluten can also trigger the symptoms of the disease. The lack of an accurate marker for monitoring compliance with the GFD is still an unresolved issue and is particularly difficult in the case of minor dietary transgressions (Fernandez-Calle et al., 1993, Gut, 34:774-777). There is no way to demonstrate gluten intake and thereby avoid possible harmful consequences. In fact, the consequences of dietary transgressions can only be measured by observing mucosal inflammation and/or villous atrophy for which intestinal biopsies would have to be performed and, as a result, the patient would have to be anesthetised with the possible consequences that this may have.
Control of anti-tTG has been proposed as a marker to assess the strict compliance with the GFD. However the effectiveness of this marker to control the intake of gluten is not yet clear (Tack et al., 2010, Gastroenterol Hepatol., 7:204-213). Other markers have been proposed for monitoring the diet, such as permeability test (Duerksen et al., 2005) or fecal calprotectin (Ertekin et al., 2010, J Clin Gastroenterol., 44:544-546). These methods can demonstrate the presence of inflammatory processes, so that if the values of these markers are altered it can be a result of infectious diseases, inflammatory bowel diseases or allergy processes, meaning that they do not need to be a measure of the direct intake of gluten. Therefore, there is no effective method to verify that the celiac patient is performing a GFD or to eliminate the possibility that the refractory CD symptoms are due to a hypersensitive intolerance to gluten traces associated with unintentional exposure to toxic cereals.
Compliance with the diet assessed by interview has been suggested as a marker of CD control for its low cost, non-invasiveness, and its proven correlation with intestinal damage. However, GFD involves numerous restrictions for patients because of its social and economic implications. Additionally, a gluten-free diet is difficult to maintain due to the ubiquity of gluten in foods, educational misinformation, changes in food labeling and possible cross-contamination in food (Bethune et al., 2009, Chem Biol, 16:868-881; Selimoglu and Karabiter, 2010, J Clin Gastroenterol., 44:4-8). Moreover, certain lifestyles and some sectors of the population make difficult, to some extent, compliance with the GFD. Furthermore there is no alternative to patient interviews to know how reliable the results of these clinical trials are.
A more direct measure of the ingestion of gluten could provide critical information about the patient: the detection of infringements of the GFD before anatomical damage, inadvertent consumption detection, the accuracy assessment of the adherence to treatment in the initial period after diagnosis when patients are less familiar with the diet, etc. providing an easy and reliable confirmation of the results obtained. Therefore, a sensitive and reliable marker to monitor and detect gluten intake could be a useful tool for the proper compliance with the GFD and probably for an accurate diagnosis of refractory CD.
Monoclonal antibodies (moAbs) G12 and A1 obtained against the main immunogenic epitope of α-gliadin have demonstrated to be very useful in the detection of toxic peptides in food samples as well as in clinical research of gluten enzymatic detoxification (Morón et al., 2008, Am J Clin Nutr., 87:405-414; Morón et al., 2008, PLoS ONE, 3: e2294; Ehren et al., 2009, PLoS ONE, 4: e6313, Alvine Pharmaceuticals, Inc., Biomedal SL). The sensitivity and specificity of the monoclonal antibodies and their ability to recognize peptides resistant to gastrointestinal digestion could make them ideal for monitoring immunotoxic gluten peptides obtained after intestinal digestion in human samples. Recognition epitopes from moAb G12, QP(Q/E)LP(Y/F), are present in major peptides described recently in a high throughput screening performed with 2,700 peptides from prolamins of different cereals (Tye-Din and al., 2010, Sci Transl Med 2:41 RA51). Undigested peptide fragments from gluten intake that are not absorbed could be recovered from the feces, which would demonstrate gluten intake by the individual.
In this patent, we have evaluated the feasibility of monitoring gluten, intact and digested, in the feces by the detection of epitopes associated with the 33-mer peptide, which could be used in clinical studies and dietary monitoring, as well as in the diagnosis of refractory CD.